MUC5AC gene. This mucin species is the major mucus glycoprotein present in the secreted gel-forming mucus. The level of gene expression caused by quercetin or with dexamethasone, if caged into polyP NP, is significantly higher compared to the individual drugs alone. Both quercetin and dexamethasone did not impair the growth-supporting effect of polyP on A549 cells even at concentrations of quercetin which are cytotoxic for the cells. A possible mechanism of the effects of the two drugs together with polyP on mucin expression is proposed based on the scavenging of free oxygen species and the generation of ADP/ATP from the polyP, which is needed for the organization of the protective mucin-based mucus layer.Inorganic polyphosphate (polyP) is a widely distributed polymer found from bacteria to animals, including marine species. This polymer exhibits morphogenetic as well as antiviral activity and releases metabolic energy after enzymatic hydrolysis also in human cells. In the pathogenesis of the coronavirus disease 2019 (COVID-19), the platelets are at the frontline of this syndrome. Platelets release a set of molecules, among them polyP. In addition, the production of airway mucus, the first line of body defense, is impaired in those patients. Therefore, in this study, amorphous nanoparticles of the magnesium salt of polyP (Mg-polyP-NP), matching the size of the coronavirus SARS-CoV-2, were prepared and loaded with the secondary plant metabolite quercetin or with dexamethasone to study their effects on the respiratory epithelium using human alveolar basal epithelial A549 cells as a model. The results revealed that both compounds embedded into the polyP nanoparticles significantly increased the steady-state-expression of the  On the cellular level, both macrophages and dendritic cells are the prime sensors of fungi, bacteria, and viruses that cause infection in humans. They are the starting points from where the innate as well as the adaptive immune defense and boosting machineries start . The innAcute respiratory infections (ARIs) are prevalent worldwide and together with diarrhea, are the leading cause of death, especially in developing countries . FocusinN- and C-terminal regions to a close-meshed, often gelatinous network.The respiratory viruses have to reach the epithelial cells via the bulky mucus layer, which is the first physical barrier that these viruses have to overcome. The epithelia of the different compartments of the upper respiratory tract are composed of specialized cells, the goblet cells, the basal cells as well as the ciliated cells and non-ciliated cells . These cMUC5AC, as well as two non-polymeric glycoproteins. The gel-forming mucins are layered outside of the epithelial cells, the periciliary layer, and form the epithelial lining fluid within the mucus layer. The directed motion of this layer is driven by the cilia protruding from the ciliated cells; the flow velocity of the mucus is 4–10 mm/min , and 1254 cm−1 for νas [(PO2)−]  . The speethasone . The spesone -d-glucose, might be a suitable way to follow [The present study shows that the cytotoxic activity of quercetin, above a concentration of 0.3 µg mLin genes . Parallein genes . It can in genes , includiin genes . Mg2+ iouercetin . PolyP r polymer . Taken t polymer . Again, o follow .The medical value of both compounds, dexamethasone and quercetin, is well established. In the present study, it is underlined that if the compounds are embedded into the polyP scaffold, a 50% release is measured after ~24 h. This delayed release is especially favorable for the growth-reducing function of quercetin, allowing an exposure of this compound for a longer period at biologically preferable concentrations.A further notable aspect of the present data is that the polyP NP undergo together with mucin a coacervate-like transformation. This observation is remarkable since the coacervate form of polyP has been shown to be the biologically active form of the polymer . Moreovei units (polyP40) came from the Chemische Fabrik Budenheim . Quercetin was purchased , similar to dexamethasone 21-phosphate disodium salt , mucin from bovine submaxillary glands (type I-S) , thiazolyl blue tetrazolium bromide  and Ham’s F-12K (Kaighn’s) medium .Na-polyphosphate (Na-polyP) with an average chain length of 40 P2 and Na-polyP (based on phosphate) was used and the pH was adjusted to 10. For the reaction, 1 g of Na-polyP was dissolved in 100 mL of distilled water and 3.86 g of MgCl2∙6H2O  were dissolved in 100 mL. The MgCl2 solution was slowly and dropwise added to the polyP solution during a 60 min period of time. The pH was adjusted close to 10 (with NaOH). After stirring for 12 h, the nanoparticles (NP) were collected by filtration, washed twice with ethanol and then three-times with water and dried at 50 °C. The particles were collected; “Mg-polyP-NP”.Amorphous Mg-polyP nanoparticles (Mg-polyP-NP) were prepared as described ,76, with2 and processed as described. Finally the particles were collected, washed three times with water, and freeze-dried; “Mg-polyP/D-NP”.For loading of the particles with dexamethasone the corticosteroid was added to polyP at a mass concentrations of 5 wt% with respect to Na-polyP . Dexamet−1). The release kinetics from the NP was measured using 20 mg of “Mg-polyP/D-NP”, as described [The encapsulation efficiency of dexamethasone in the “Mg-polyP-NP” was determined spectrophotometrically by ultraviolet–visible spectroscopy . A calibration curve was established using DEX-P, as described , and theescribed . The par−1. In this step, 10 mL of the QCT solution was supplemented with 100 mg of “Mg-polyP-NP” and stirred for 60 min. Then, the particles were collected, washed with water and used for the experiments; “Mg-polyP/QCT-NP”.For the preparation of quercetin-polyP nanoparticles (Mg-polyP/QCT-NP) the “Mg-polyP-NP” were, after preparation, washed in an ethanolic QCT solution of 0.3 mg mLIn order to determine the QCT content in “Mg-polyP/QCT-NP”, the particles were suspended in citric acid (at pH 3.0) and incubated for 12 h at 37 °C. Then, the supernatant was collected, and the absorbance was measured at 366 nm ,47,79,80−1 and 1.49 g mL−1 was collected. Where indicated, an aliquot of 2 mg was subjected to 1 mL of 0.25 M NaCl and supplemented with 10 mg of “Mg-polyP-NP”.Commercial mucin (from bovine submaxillary glands) was purified as described . The fraThe release kinetics of QCT from “Mg-polyP/QCT-NP” was determined in medium/serum. An aliquot of 10 mg of the particles was suspended in 5 mL of medium/serum; then, after the indicated time, the level of QCT was determined in the supernatant spectrophotometrically.The scanning electron microscopic (SEM) images were performed with a HITACHI SU8000 microscope .The Zetasizer Nano ZS90  was used to determine the size of the particles by light scattering ,83. The Fourier transformed infrared spectroscopy (FTIR) was carried out with an ATR -FTIR spectroscope/Varian 660-IR spectrometer , fitted with a Golden Gate ATR unit . The samples were pressed to small discs and subjected to the determination.2 in air (37 °C) as described [A549 cells , a human lung (carcinoma) line, were cultivated in Ham’s F-12K (Kaighn’s) medium, supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 4 mM glutamine. The cells were incubated in 96-well plates or 24-well plates in a humidified atmosphere of 5% COescribed . Every 32+  was added to the assays in order to compensate for the complexation potential of the polyanionic polymer [The incubation with polyP, both Na-polyP or the indicated Mg-polyP formulation, either the one-component “Mg-polyP-NP”, or this preparation together with dexamethasone “Mg-polyP/D-NP” or QCT “Mg-polyP/QCT-NP” at the indicated concentration was added. In the series with Na-polyP additional Mg polymer .−1; 2 h) and subsequently with 20% SDS in 50% dimethyl-formamide for 24 h [The viability kinetics of the cells was assessed by the determination of the metabolic activity, using the MTT assay . The incfor 24 h . The for3 cells mL−1 medium/serum and incubated for 3 and 6 d, respectively. Then, the cells were harvested, and RNA was extracted and used for the qRT-PCR reaction. The following primer pairs were used: for human MUC5AC [GAPDH  with the primer pair Fwd: 5′-ACTTTGTGAAGCTCATTTCC-3′ and Rev: 5′-TTGCTGGGGCTGGTGGTCCA-3′. The amplifications were performed in triplicate in an iCycler , and the mean Ct values and the efficiencies were calculated applying the iCycler software (Bio-Rad) [A detailed description of the qRT-PCR procedure has been given before . The A54n MUC5AC  (U06711)Bio-Rad) ; the estMUC5AC antibodies ; 1:1000 dilution in blocking reagent. Subsequently the samples were reacted with goat anti-rabbit IgG secondary antibodies . The specimens were inspected with an Olympus AHBT3 microscope.After termination of the experiments after a 3 d incubation period, the A549 cells were washed in PBS, fixed and permeabilized with methanol/EGTA for 10 min . Then, tt test was applied to perform comparisons between two groups. Usually, the average values and σ originated from at least three to six independent experiments. Values of p < 0.05 were considered statistically significant (*). The calculations were performed with the GraphPad Prism 7.0 software .The skewness of the non-Gaussian distribution curves of the NP was evaluated as published ,83,90. TMUC5AC. One common mode of action might be the fact that all three components are able to scavenge free oxygen species. This property has also been proposed for phosphate that is released during the ALP-mediated enzymatic cleavage of polyP; at least some metal salts (Mn2+) of phosphate may inactivate oxygen radicals [+ oxidoreductase seems to involve the formation of a metaphosphate intermediate [The present study shows that caged NP formed of polyP with dexamethasone and QCT display morphogenetic activity and cause an increased expression of mucin, as demonstrated here with radicals . It mighradicals . Phosphoradicals  and evenradicals , but it rmediate , as proprmediate . Besidesrmediate  also hasrmediate , as show


A high density of Granzyme B, FOXP3, CD68, CD206, PD-1, and CTLA-4 was associated with better disease-specific survival (DSS). The patients with diffuse PD-L1 tumor cell expression had worse prognoses than those with marginal or negative PD-L1 expression. Four immunophenotypes were identified by unsupervised clustering analysis, based on certain immune markers, which were associated with DSS and lymph node metastasis (LNM) in peSCC. There was no significant relationship between the immunophenotypes and high-risk human papillomavirus (hrHPV) infection. However, the hrHPV–positive peSCC exhibited a higher density of stromal Granzyme B and intratumoral PD-1 than the hrHPV–negative tumors . In conclusion, the immunophenotypes of peSCC were of great value in predicting LNM and prognosis, and may provide support for clinical stratification management and immunotherapy intervention.The tumor immune microenvironment (TIME) plays an important role in penile squamous cell carcinoma (peSCC) pathogenesis. Here, the immunophenotype of the TIME in peSCC was determined by integrating the expression patterns of immune checkpoints (programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1), cytotoxic T lymphocyte antigen 4 (CTLA-4), and Siglec-15) and the components of tumor-infiltrating lymphocytes, including CD8 Indeed, the c-statistic was 0.861 for the model with T stage, N stage, and hrHPV, and was 0.875 after incorporating the immunophenotypes. Thus, the identification of the immunophenotypes is of great significance for explaining peSCC survival.We also analyzed the prognostic impact of the immunophenotypes while considering other prognostic factors. Univariate factors associated with the DSS were identified by a multivariate Cox proportional hazard analysis, as shown in We further investigated the association of the immunophenotypes with well-known clinicopathological features  in peSCC, as shown in p = 0.001), as shown in p = 0.021), as shown in No statistically significant difference among the four groups for hrHPV infection was found. Cluster A had a greater proportion of advanced T stages (T3 or T4) than the other three clusters ). The results indicate that patients in cluster A were frequently accompanied with advanced T and N stages.In multivariable analysis, as shown in The tumor immune microenvironment plays an important role in the pathogenesis of peSCC. This is the first study, to our knowledge, to report the expression of GrB, PD-1, CTLA-4, and Siglec-15 in peSCC and to propose four immunophenotypes with relevance to the prognosis and LNM, based on multiple immune parameters.+ CTLs are primarily responsible for killing tumor cells, while the increase in Tregs is thought to inhibit host immunity and promote tumor progression. In addition, GrB is a cytotoxic molecule secreted from activated CTLs and natural killer cells that induce neoplastic cell apoptosis [+, CTLA-4+ T cells, and FOXP3+ Tregs was counter-intuitive. The results contrasted with previous studies in peSCC and other tumor types [CD8poptosis . As expepoptosis . Despiteor types .+ or PD-1+ cells is limited. CTLA-4 exhibited a linear positive correlation with CD8+ CTLs and FOXP3+ Tregs, due to CTLA-4 being predominantly expressed in FOXP3+ Tregs and newly activated T cells [+ Tregs were observed to be positively related to CD8+/GrB+ CTLs, and PD-1+ and CTLA4+ T cells, suggesting that the upregulation of FOXP3, PD-1, and the CTLA-4 expression of T cells could be the result of increased immune infiltration.There may be several reasons for this discrepancy. The expression of CTLA-4 and PD-1 reflects the activation of T cells, but only indicates that T cells have received an immunosuppressive signal, not that the T cells must be depleted and dysfunctional. Our results indicate that, despite the varying functional status, grouping CTLA-4 T cells . FOXP3+ + CTLs infiltration. This indicated that the progression and prognosis of peSCC may result from a combination of immune factors.We found that patients with diffuse PD-L1 tumor expression had significantly poorer prognoses than those with marginal expression. The expression of marginal PD-L1 may be induced by extrinsic factors, such as interferon gamma (IFNγ), tumor necrosis factor α (TNFα), and interleukin-1β (IL-1β), produced locally by activated T lymphocytes in the stroma ,27. In cHence, to provide a more comprehensive analysis of the TIME in peSCC, immunophenotypes based on multiple immune factors become necessary. Through unsupervised clustering, we identified four clusters of patients with peSCC. These immunophenotypes: (i) were related to varying immune factor infiltrations and specific tumor immune microenvironments; (ii) provided an independent prognostic indicator; (iii) provided a significant predictor for LNM.Immune cluster A was characterized by a deficiency of TILs and TAMs in either the tumor or the stroma with no PD-L1 expression. Therefore, this group was called “immune ignorance” or “cold tumors” immunophenotype. Immune ignorance was associated with an advanced T stage, LNM, and had the poorest disease prognosis among the TIME subgroups. This may be attributed to the lack of the expression of activation markers and rejection by tumors, where adaptive immunity cannot recognize or respond to pathogens or malignant tumors . Studies+ cytotoxic T-cell infiltration with high PD-L1 expression; thus, this was named the “immune inflammatory” immunophenotype. CD8+, TILs, and PD-L1 often showed a positive correlation, indicating that an adaptive immune resistance mechanism may be occurring. Deng et al. found that CD8+ TILs could induce PD-L1 expression in tumors through the production of IFNγ [The most representative pathologic feature of cluster B was the high levels of intratumoral CD8 of IFNγ . This su of IFNγ . Therefo+ macrophages in the tumor core, was termed as the “immune exclusion” immunophenotype. This group was found to be related to LNM and to a poor clinical prognosis in peSCC. Interestingly, the high numbers of CD68+ and CD206+ TAMs were associated with improved prognoses in our cohort. In contrast, several other studies found that a high level of CD206+ TAMs was positively correlated with shorter survival among HNSCCs and hepatic carcinoma [+ macrophages were a potential anti-tumor therapeutic target [Immune cluster C, which was broadly populated with immune cells but relatively void of CD206arcinoma ,31. Sevearcinoma . CD206-ec target .Immune cluster D had moderate levels of lymphocyte and macrophage infiltration with high stroma Siglec-15 expression and was named the “immune tolerance” immunophenotype. This group comprised relatively early tumor stages and was associated with good prognoses. Siglec-15 is widely upregulated on various tumor cells and tumor-infiltrating myeloid cells, as it is normally only expressed on certain myeloid cells and its expression is mutually exclusive to PD-L1 in non-small-cell lung carcinomas (NSCLCs). Siglec-15 was negatively regulated by macrophage colony-stimulating factor (CSF) and IFNγ, thus inhibiting antigen-specific T cell responses and promote tumor growth . It is c+ peSCC had a higher density of stroma GrB and intratumoral PD-1 positive T lymphocytes. The tumor microenvironment of hrHPV+ peSCC was somehow different from that of hrHPV− cancer. HPV–positive HNSCCs were much more strongly immune-infiltrated compared with HPV–negative tumors, with higher levels of T cell infiltration and overall immune cell infiltration [+ CTLs, HPV+ tumors harbored higher levels of immune activation—specifically perforin, Granzyme A, and Granzyme B expression. Interestingly, the better outcomes seen in HPV+ tumors may be partially related to their higher immune infiltration, as we observed. This may be explained by the highly immunogenic property of hrHPV, which could recruit immune effector cells, and upregulate the PD-1 and CTLA-4 immunosuppressive pathways [+ peSCC, wherein a high tumoral HPV-antigen load resulted in the high expression of PD-1 on T-cells. However, the mechanisms by which HPV infection affects the immune checkpoint pathways are yet to be fully defined. Although we found no difference in the hrHPV infection rates among the four immunophenotypes, hrHPVltration . Consistpathways . Our finThere were some limitations worth addressing in the present study. First, it was a single-center retrospective study. In some cases, several immunohistochemical tests were not available due to insufficient tissue materials. Second, we chose CD68 and CD206 to label macrophages and M2 macrophages, respectively. There are also alternative immune molecular markers, such as CD163, CD11, etc., for labeling M2 macrophages, which may lead to some differences in the results. Third, we used digital high-resolution images and professional computer software to evaluate immune markers objectively and quantitatively, thus limiting the subjectivity of the observer. However, there was no uniform standard for the cutoff value of immune markers, such as the mean or median, and we used the cutoff value for the DSS as the cutoff value. Fourth, we did not perform multiple immunofluorescence tests on all the samples to label and locate specific immune cells in the TIME. External validation is required in other populations to confirm these study findings.The study included all men with peSCC admitted to the Sun Yat-sen University Cancer Center (SYSUCC)  between January 1999 and December 2013. One hundred and seventy-eight consecutive patients diagnosed with peSCC were eligible with the following criteria: (1) histologically confirmed diagnosis of invasive peSCC; (2) sufficient formalin-fixed, paraffin-embedded (FFPE) block with available primary tumor tissue for this study; (3) no other tumor occurrences; (4) availability of the patient’s complete clinical history and pathological diagnostic data.For each patient, their clinicopathological information  system), presence of lymphovascular and nerve invasion, and necrosis ratio) and follow-up data were retrospectively reviewed. The tumor histology subtypes were classified as HPV-related or non-HPV-related carcinomas in whole tissue sections, according to the World Health Organization (WHO) 2016 classification of peSCC . The stuThe hrHPV status was determined on FFPE tissue samples using real-time polymerase chain reaction (PCR) with a Hybribio Assay  that detected the 23 HPV types . The kitThe representative FFPE tissue specimens were sectioned continuously at four micrometers thick and stained for immunohistochemistry. Staining was performed following the manufacturer protocols. The details of the different IHC staining processes are summarized in For CD8, GrB, FoxP3, CD68, CD206, PD-1, and CTLA-4 analysis, we used the Aperio ImageScope  to randomly select three peripheral and three central tumor focus fields magnified by 200× in each sample. Necrotic areas representing nonspecific inflammatory infiltration were excluded. The inForm 2.1 Image Analysis Software (Mantra Software/PerkinElmer) was used to analyze the captured images, which included the intratumoral and peritumoral stromal regions, as shown in + tissues for CD8/PD-L1 and CD68/PD-L1 dual staining. PD-L1 staining was first performed using the avidin–biotin horseradish peroxidase (HRP) technique. After color development, the 3,3′Diaminobenzidine tetrahydrochloride substrate (DAB) was removed, and the sections were washed with water and phosphate-buffered saline (PBS). Then, the CTLs marker, CD8, or TAMs marker, CD68, was labelled using streptavidin–biotin–alkaline phosphatase complex  system with vector red as a chromogen.For PD-L1 staining, >1% tumor cell membranous staining was considered as positive, and the PD-L1 expression of immune cells in the stroma was binarily scored (negative or positive) . We evalFor estimating the Siglec-15 expression, three high fields  containing the most stained Siglec-15 (‘hotspots’) were assessed using an Olympus BX53 microscope . Then, the mean numbers of intratumoral and stromal positive cells were counted in the hotspots. The tumor and stroma were separately scored as follows: 0—<1 cells/high magnification field; 1—1 to 5 cells/high magnification field; 2—5 to 20 cells/high magnification field; 3—20 to 50 cells/high magnification field; 4—>50 cells/high magnification field. All the results were evaluated and checked by two pathologists (Chu CB and Lu JL) who were blinded to the patients’ clinical data.t-test or the Kruskal–Wallis test for continuous variables.Statistical analyses were performed using SPSS  and R software . The figures were created using R software. The normality and log-normality of the distributions of continuous variables were examined using the Shapiro–Wilk test. The immune cell counts with a lognormal distribution were transformed using a natural logarithm (ln). The optimal cutoff points of the expressions of CD8, GrB, FOXP3, CD68, CD163, PD-1, and CTLA-4 were determined by the “surv_cutpoint” function of the “survminer” R package. Accordingly, the patients were divided into a low- or high-expression group, with the optimal cutoff point for each immune marker serving as the cut-off value. Comparisons between groups were performed using Fisher’s exact test or the Chi-square test for the categorical variables and Student’s The association between different variables was performed with the Spearman rank correlation analysis. The package ggcorrplot was used for visualizations. To consider the complexity of the TIME of a large number of immune markers, we used a statistical method of unsupervised clustering, as previously used in genomic research. The clusters of the immunophenotypes were performed with the help of the R package pheatmap v1.0.12, using correlation as the clustering distance and ward.D as the linkage. Clusters were identified using the cutree function. To determine the optimal number of clusters, we performed elbow analysis of the KMeans using the “factoextra” R package.p < 0.10 in the univariate analysis were selected for the multivariable backward stepwise Cox regression to build prognostic models. The c-statistic was used to assess the models’ goodness of fit. The predictive value of LNM was investigated using a logistic regression analysis. The sensitivity and specificity of the multivariate logistic regression models were measured using the area under the receiver operating characteristic (ROC) curve (AUC). The results were considered significant for p-values < 0.05.The disease-specific survival (DSS) was calculated from the date of diagnosis until disease-caused death or the end of the follow-up. The Kaplan–Meier method and the log-rank test were used in the univariate analysis. Variables with In conclusion, peSCC grows in a very complex microenvironment, which can affect tumor progression and prognosis. We thereby proposed four immune-related clusters of the TIME in peSCC, which can improve prognosis stratification and provide new insights for the development of therapeutic treatments in the future.


The ∆f values for the p-MIP (MIP on gold pinholes) and s-MIP films (MIP on silica skeleton surface) were obtained with the ∆f value of −199 ± 4.9 Hz and −376 ± 19.1 Hz, respectively, whereas for p-/s-NIP films, the ∆f values were observed to be −115 ± 19.2 Hz and −174 ± 5.8 Hz by the influence of non-specific adsorption on the surface of the films. Additionally, the imprinting factor (IF) appeared to be 1.72 for p-MIP film and 2.15 for s-MIP film, and the limits of quantitation (LOQ) and detection (LOD) were 54.924 and 18.125 nM (p-MIP film) and 38.419 and 12.678 nM (s-MIP film), respectively. Using the Freundlich isotherm model, the binding affinity of the BPA-imprinted films was evaluated. This was measured in an aqueous solution of BPA whose concentration ranged between 45 and 225 nM. It was found that the p-MIP film (m = 0.39) was relatively more heterogeneous than the s-MIP film (m = 0.33), both of which were obtained from the slope of the linear regressions. Finally, the selectivity of the MIP-QCM sensors for BPA detection was determined by measuring the effect of other analogous chemicals, such as bisphenol F (BPF), bisphenol AP (BPAP), and bisphenol B (BPB), in aqueous solutions. The selectivity coefficients (k*) of the two MIP films had ~1.9 for the p-MIP and ~2.3 for the s-MIP films, respectively. The results reveal that, with respect to signal amplification of the QCM sensors, the s-MIP film has better sensing features and faster detection responses than the p-MIP film.This study successfully fabricated BPA-imprinted poly(4-vinylpyridine-co-ethylene glycol dimethacrylate) (poly(4-VP-co-EGDMA)) quartz crystal microbalance (MIP-QCM) sensors on a silica skeleton surface and gold pinholes of silica inverse opal through surface-initiated atom transfer radical polymerization (SI-ATRP). The sensing features of the two MIP films on the structured silica surface and nano-scale local gold surface were investigated by measuring the resonant frequency change (∆ Humans are exposed to high levels of toxic bisphenol A propane; BPA) through dietary contamination, inhalation, and physical contact with BPA-containing materials . This co2, and polymers and has successfully controlled graft polymerization on the surface [f) relating to the template mass loading (∆m). MIP film-modified QCM (MIP–QCM) sensors have been proven to be one of the most accurate and reliable analytical tools in use because numerous substances, such as viruses, proteins, and clinical biomarkers, including food and beverage applications, have been quantified using MIP-QCM [The novel development of molecular recognition systems with artificial receptors using an imprinting technique has been highly interesting for effective separation systems and chemical/biosensors . In the  surface ,14,15,16 surface , electro surface , and gra surface  analyses MIP-QCM .s-MIP or p-MIP), were systematically designed to investigate the effects of the surface geometry and active surface area on which MIP films were thermally grafted on the sensing behaviors. Using the two different MIP films, the sensing effects of precisely designed MIP geometry on the structured silica surface and nano-scale local gold surface of QCM sensors, associated with analyte diffusion toward structured surfaces, were investigated. For that purpose, sensing properties, such as sensitivity and selectivity, were obtained by monitoring resonant frequency change (∆f) from QCM sensors, and the Freundlich isotherm model was applied in our MIP sensing systems and evaluated binding affinity.In this study, BPA-imprinted poly(4-vinyl pyridine-co-ethylene glycol dimethacrylate) (poly(4-VP-co-EGDMA)) films were synthesized on a specific surface (silica skeleton and gold pinholes) of silica inverse opal-formed quartz crystals (QCs) using surface-initiated atom transfer radical polymerization (SI-ATRP). It was discovered that 4-VP functional monomers typically interacted with BPA in the MIP matrix through non-covalent hydrogen bonding, van der Waals force and π–π interaction . Two MIPPolystyrene (PS) latex beads , which were purchased from Alfa Aesar Co. , were served as a sacrificial monolayer mask for 2D silica inverse opal. Sodium dodecyl sulfate  was used as an anionic surfactant for a PS bead monolayer, thereby facilitating buoyancy and highly packing at the air–water interface. Hydrochloric acid  and (3-mercaptopropyl)trimethoxysilane  were used to chemically immobilize porous silica sol-gel films on 9 MHz gold-coated AT-cut Au-coated quartz crystals . Tetraethyl orthosilicate  and absolute ethanol  were supplied from Sigma-Aldrich Co., St. Louis, MO, USA. Trimethoxy(methyl)silane (TMMS) and cysteamine was purchased from Tokyo Chemical Industry Co., Tokyo, Japan. Triethylamine , 4-(dimethylamino) pyridine , 2-bromoisobutyryl bromide , and anhydrous dichloromethane  were used to immobilize the initiator for atom transfer radical polymerization (ATRP). For MIP synthesis, 4-vinylpyridine , ethylene glycol dimethacrylate , bisphenol A , and anhydrous acetonitrile  were, respectively, used as functional monomer, cross-linker, template molecule, and solvent. Copper (I) bromide  was used as the catalyst and N, N, N′, N″, N″-pentamethyldiethylenetriamine  was used as ligand. PMDETA was dried with calcium hydride (CaH2) which was obtained from Junsei Chemical Co. and distilled at low pressure. Bisphenol F (BPF), bisphenol AP (BPAP), and bisphenol B (BPB) were received from Sigma-Aldrich Co. Deionized (DI) water  and all other materials were used as received.2SO4:30% H2O2 = 3:1 vol%) for 40 min to make the surface hydrophilic. Furthermore, the PS colloidal dispersion solution (80 μL) was then distributed on the pre-treated substrate using a micropipette and spin-cast at a spinning rate of 300 rpm for 5 min using a spin-coater . Lastly, the colloids were transferred to the water surface in a plastic Petri dish and the irregular polystyrene colloid monolayer was highly ordered by adding 2 wt% SDS solution (200 μL) in drops. The monolayer was placed on a QC substrate and completely dried for a day.A hexagonal close-packed colloidal monolayer was assembled as a sacrificial mask at the air–water interface and then transferred onto QCs after dropping an SDS aqueous solution . First oA silica sol-gel precursor solution was prepared following the previously reported procedure . As the 2 gas, the QCs were immersed in an aqueous solution of 18 mM cysteamine for 4 h [2 gas, the MIP films were prepared as follows: the initiator-functionalized substrate was placed in an Erlenmeyer flask under N2 gas stream and a mixture solution containing 4-VP , EGDMA , and BPA  in anhydrous acetonitrile (25 mL) was injected into the flask, followed by the addition of the catalyst/ligand solution (14 mg CuBr and 32 μL PMDETA in 5 mL anhydrous acetonitrile). After purging with N2 gas for 10 min, the SI-ATRP reaction was carried out at 48 ± 2 °C for 24 h under gentle stirring. The MIP film was then rinsed with acetonitrile and dried with N2 gas to remove any remaining residues. Finally, the BPA molecules in the MIP film were extracted in methanol for 1 h. However, the methylation of silica surfaces and cysteamine immobilization were omitted for the s-MIP formation to make the silica surfaces active. The modification of the initiator and ATRP synthesis on the silica surfaces was followed by the aforementioned procedures. Finally, non-imprinted polymer (NIP) films were also fabricated using the same procedure but in the absence of BPA molecules.Two independent active synthesis sites (gold pinhole and silica surface) on the 2D hexagonal porous silica films were prepared for grafting the BPA-imprinted films. To make the gold pinhole active for MIP synthesis, the silica skeleton on the QCs was blocked with a methyl group by soaking in a mixture of TMMS (28.8 mL), absolute ethanol (25 mL), and 0.1 N HCl aqueous solution (25 mL) for 4 h at room temperature. After rinsing with ethanol several times and drying with N for 4 h . Subsequ for 4 h . After rf) obtained was converted to the mass of adsorbed analyte [2 ng−1), a decrease of 1 Hz in the ∆f value was almost equal to 1.07 ng in the amount of mass loaded on the total gold area (A = 0.19625 cm2) [f values of the MIP and NIP films were measured in the air before and after the BPA-extraction process using a QCA 922 quartz crystal analyzer  to determine the mass of the BPA-free polymer matrix and calculate the maximum binding capacity (Qmax).MIP/NIP film sensitivity was evaluated by monitoring in a 125 mL aqueous solution with BPA concentrations ranging from 45 to 225 nM for a 1-h rebinding process. Furthermore, the sensing response was recorded in the respective aqueous solutions containing 225 nM BPA and its analogs  to evaluate the selectivity of the QCM sensors for recognizable BPA binding sites. With the use of the Sauerbrey Equation, the average resonant frequency change  . The Δf Atomic force microscopy  and field-emission scanning electron microscopy  were used to analyze the surface topography of the MIP films on porous silica sol-gel structures.s-MIP) or the gold pinhole (p-MIP). For more clarity of the MIP formation, AFM images are shown in s-MIP film. The s-MIP film grew on the entire surface of the silica skeleton and exhibited a considerably rough surface, whereas the p-MIP film formed only near the gold pinhole area. In both MIP films, structurally recognizable functional cavities were easily generated by breaking the non-covalent bond between the BPA templates and the functional monomers in the polymer matrix. In general, structured MIP-QCM sensors could show amplified ∆f signals in a limited sensing period compared with the simple planar films because of an increased surface area to volume (S/V) ratio [A hexagonally packed PS colloidal monolayer was used as a sacrificial mask in which the silica precursor was filled into the voids of PS colloids arranged on the QCs. A well-ordered porous structure with a gold pinhole at the center of each pore was obtained after the formation of the silica sol-gel network and the removal of the PS colloid by toluene rinsing . As showV) ratio . The thif value, depending on BPA binding to accessible cavities for the two MIP films , was monitored in a 225 nM aqueous solution (125 mL) of BPA for an hour rebinding process. As shown in f values for both imprinted films increased gradually with rebinding time, indicating that the BPA molecules were predominantly diffused into the films and non-covalently bonded to functional nanocavities. The sensing signal of the s-MIP film was amplified  compared with the ∆f value for the p-MIP film (−199 ± 4.9 Hz). When the ∆f value of each film was converted into the appropriate Qe values , the Qe values could be comparatively evaluated, resulting in a value of 134 ± 3.3 mg/g for the p-MIP film and 73 ± 3.7 mg/g for the s-MIP film (max) of both MIP films was calculated by mass difference before and after BPA removal from the MIP films. Although the MIP mass grown on the silica surface was much larger than that on the gold pinhole, the p-MIP film with Qmax (170 mg/g) film had more recognizable cavities than the s-MIP film (Qmax = 115.4 mg/g), owing to the effects of film growth and thickness. According to the Qmax and Qe values, 78.7% of the binding sites in the polymer matrix were filled with BPA molecules on the p-MIP film, whereas the corresponding value for the s-MIP film was 63.7% for a 1-h rebinding process because of the thicker MIP film forming on the silica surface. For p-/s-NIP films, the ∆f values were obtained in a relatively lower range between −115 ± 19.2 Hz and −174 ± 5.8 Hz (n = 3), owing to the influence of non-specific adsorption on the surface of the NIP films in the absence of functional cavities —i.e., ∆fMIP/∆fNIP [p-MIP and s-MIP films, respectively.The ∆MIP film . The maxcavities . The ∆f IP/∆fNIP —was 1.72f values in aqueous solutions of BPA with the concentration ranging between 45 and 225 nM  and LOD = 3.3 (S/m), where S is the standard deviation of the intercept and m is the slope of the regression line, the limits of quantitation (LOQ) and detection (LOD) were calculated from linear calibration curves for the two MIP films in p-MIP film) and 38.419 and 12.678 nM (s-MIP film), respectively. These results indicate that the s-MIP film maintained relatively lower quantitative limits than the p-MIP film. Similarly, the two NIP films were also investigated, as shown in The BPA binding on the two different MIP films was measured using the ∆d 225 nM . As showB) were recalculated from the sensing response (∆f) of the MIP-QCM sensors, and the concentration of the free analyte (F) in the remaining solution after a 1-h rebinding process was gained from the initial concentration (C0) and the bonded BPA mass. As shown in m and a) of the Freundlich model, as shown in Equation (1), indicate the Freundlich heterogeneity index and the pre-exponential factor including the Freundlich capacity (NT) and average affinity (K0) parameters, respectively.For the BPA-imprinted QCM sensors, the Freundlich isotherm model was applied to assess the binding behaviors on the heterogeneous MIP films within the examined concentration range ,35. The p-MIP film (m = 0.39) was relatively more heterogeneous than the s-MIP film (m = 0.33), which was obtained from the slope of the linear regressions. Approximations of the exponentially decaying distribution, in association with the continuous affinity distribution expression for the N and K values, could be predicted by the Freundlich model using the Equation (2).K is the affinity constant in 1/M and N(K) is the number of binding sites for a given affinity in μmol/g [N(K)–log K plot   ) of the two MIP films increased in the following order: 1.897 (BPF) < 1.892 (BPAP) < 1.912 (BPB) for the p-MIP film and 2.267 (BPF) < 2.293 (BPAP) < 2.300 (BPB) for the s-MIP film. Thus, the s-MIP film-based QCM sensor showed more enhanced selective recognition, which resulted in remarkable signal amplification.The specific selectivity of the two BPA-imprinted MIP films was investigated by comparing the sensing response of similar chemical compounds a. The ∆f or BPB) . As shows-MIP and p-MIP films) were successfully fabricated on silica skeleton surfaces and gold pinholes of silica inverse opal using the SI-ATRP synthesis method. The sensing features  of the two MIP films on large surface areas were investigated by measuring the ∆f values of the QCM sensors. The ∆f values for the p-MIP and s-MIP films were amplified , whereas for p-/s-NIP films, the ∆f values were obtained in a relatively lower range, between −115 ± 19.2 and −174 ± 5.8 Hz, by the influence of non-specific adsorption on the surface of the films. In addition, the IF for the p-MIP and s-MIP films was 1.72 and 2.15, respectively. Based on the ∆f value obtained, the LOQ and LOD values were calculated to be 54.924 and 18.125 nM (p-MIP film) and 38.419 and 12.678 nM (s-MIP film), respectively. Finally, the selectivity coefficients (k*) were calculated to be in the range 1.897–1.912 for the p-MIP film and 2.267–2.300 for the s-MIP film. From all the results, with respect to signal amplification of the QCM sensors, the s-MIP film showed better sensing features and faster detection response than the p-MIP film. Moreover, the Qe value of the p-MIP film was greater than that of the s-MIP film because of the difference in the total MIP mass grown from different active substrate areas.Two BPA-imprinted poly(4-VP-co-EGDMA) (i.e.,


The estimated 24-h UK using the K-method and T-method had correlation coefficients of 0.36 and 0.39  and ICCs of 0.31 and 0.27 . The mean bias for the K-method in estimating the 24-h UNa and UK were the least biased among these methods. The bias between the 24-h urine Na/K ratio and the spot urinary Na/K ratio by the Bland–Altman method was −0.22. These methods for estimating the 24-h UNa and UK from SU were inadequate at the population level in Zhejiang Province, although the K-method showed the least bias among these methods. The spot urine Na/K ratio may be a useful and alternative method for 24-h urine collection for the estimation of the urinary Na/K ratio in the Chinese population.The commonly used methods of estimating the 24-h urinary sodium (UNa) and urinary potassium (UK) from spot urine (SU) are the Kawasaki method (K-method), INTERSALT method (I-method), and Tanaka method (T-method), but the method that is suitable for use in the general Chinese population is still uncertain. We aimed to assess and validate these methods in estimating the 24-h UNa and UK using SU samples in Chinese adults. We studied 1428 individuals aged 18–69 years using SU and 24-h urine samples. For the K-method, I-method, and T-method, the Pearson correlation coefficients of the 24-h UNa were 0.35, 0.35, and 0.33 (all  Excess sodium intake increases the risk for high blood pressure and is a leading risk factor for cardiovascular disease , 2. ReduHowever, 24-h urine collection is very inconvenient in large epidemiological surveys in free-living populations . LimitatBased on the above considerations, the spot urine (SU) method is an alternative in estimating daily urine sodium and potassium excretion that has attracted increasing attention. Several studies have developed equations for facile and precise 24-h urinary sodium (UNa) and urinary potassium (UK) estimation based on SU. The mostly commonly used methods are the Kawasaki method (K-method) , INTERSASome studies also recently found that the Na/K ratio from repeated casual urine specimens is useful for estimating the 24-h Na/K ratio in normotensive and hypertensive Japanese individuals , 12. TheThe aim of this study was to validate and assess these methods to predict the 24-h UNa, UK, and Na/K ratio using SU samples among the general Chinese population.This study is based on the data obtained from the baseline survey of the Zhejiang salt reduction and Hypertension project, which was conducted at five sites in Zhejiang Province in 2016. This cross-sectional survey used a four-stage stratified sampling method to select a representative sample of the general adult population aged 18–69 years. A total of 7500 participants aged 18–69 years were selected among five counties and districts. All participants were invited to participate in a close-ended questionnaire, and a physical examination was conducted by trained public health staff. A subsample of 1500 participants were randomly selected and invited to provide 24-h urine collections and morning SU.Our study was conducted according to the Declaration of Helsinki guidelines and any procedures involving human subjects were approved by the ethics committee of the National Center for Chronic and Noncommunicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention. All study participants provided written informed consent.Before the investigation began, health professionals suggested that the subjects should not change their daily life habits. The participants were given a 50 ml cup and a 10 ml tube to collect their morning fasting urine at home and carried the SU to the health professional. Then, the health professional carefully explained to the subjects the key point of the 24-h urine collection and asked the subjects to correctly repeat the process. The participants were given a standard plastic container containing ~1.0 g boric acid as a preservative for the 24-h urine. The first urine was discarded before collection. The supervising health professional recorded the start and end times of each 24-h urine collection and determined the exact duration of the collection.Each participant was interviewed using a standard questionnaire to assess the completeness of the 24-h urine collection. The total volume of the collection was measured by a laboratory technician, and the urine aliquots were frozen at −20 °C and shipped to a certified laboratory . UNa and UK were measured with an ion-selective electrode method using the Abbott Architect C16000 autoanalyzer. Urinary creatinine was measured with the picric acid method using the Cobas C501 analyzer. The completeness of 24-h urine collection was defined by urine volume and urinary creatinine. A 24-h urinary volume less than 500 ml or a 24-h urinary creatinine volume that was ±2 standard deviations (SD) outside of the sex-specific mean were considered incomplete .2. Blood pressure was measured three times by an electronic sphygmomanometer , and the average of the three measures was calculated and used for the analyses.We collected the subjects' sociodemographic characteristics and several lifestyle factors using face-to-face interviews by specially trained research staff and employing a standard questionnaire. The physical measurements included height, weight, waist circumference, and blood pressure. The height and weight (without shoes) was obtained using standardized techniques and calibrated equipment. The body-mass index formula was as follows: weight (kg)/height (m)We used the K-method, I-method, and T-method to estimate 24-h UNa using the SU sample. The INTERSALT formula was not designed to estimate the UK excretion, and therefore, we used the K-method and T-method to estimate 24-h UK excretion. All of the formulas’ expressions are available in Table t-test. We used the Pearson correlation coefficient and intraclass correlation coefficients (ICCs) to assess the three methods between estimated and measured 24-h UNa (UK) and visualized the results using scatter plots. The Bland–Altman method was used to evaluate the agreement between the estimated and measured 24-h UNa (UK) [p value < 0.05 was considered significant.Data are expressed as either the means and SD for continuous variables or as percentages for categorical variables. The estimated 24-h UNa and UK were calculated using these formulas in Table UNa (UK) . The PeaWe excluded six participants with missing physical examinations or other variables of interest and 66 participants with incomplete 24-h urine collection. Finally, 1428 participants were included in the final analysis. The characteristics of the subjects are shown by sex in Table t = 8.38, p < 0.01). However, the mean estimated 24-h UNa using the I-method and T-method was lower than the measured 24-h UNa .The differences between the measured and estimated 24-h UNa are presented in Table t = 5.40, p < 0.01). However, the mean estimated 24-h UK using the T-method was lower than the measured 24-h UK .The differences between measured and estimated 24-h UK are presented in Table p < 0.01)  . The ICCs of 24-h UK were 0.31 for the K-method and 0.27 for the T-method .The Pearson correlation coefficients between the estimated and measured 24-h UNa using the three methods were all low Fig. . The K-m01) Fig. . The Pea01) Fig. . The ICCWe used the Bland–Altman method to assess the agreement between the estimated and measured 24-h UNa (UK) Fig. . The resP < 0.01). The correlation coefficient for the Na/K ratio between the values for SU and the corresponding values for 24-h urine was 0.43 , which was the highest compared with corresponding R-values for spot and 24-h UNa or UK  participants were taking an antihypertensive medication. After excluding the 202 individuals receiving antihypertensive medication, the results were similar . Compared with the results of previous studies, our results are similar or much lower [We adjusted the process based on the creatinine concentration in all three methods because of its consistent excretion in urine –19. If uch lower –23. For ch lower . Accordich lower . Our stuMany studies have suggested using the SU sample to estimate 24-h UNa as a substitute for the 24-h urine collection method . HoweverOur study was similar to certain other studies in Chinese adults. The study in Shaanxi Province, China, also found significant biases for these three methods in estimating 24-h UNa, and the least bias and highest agreement occurred for the K-method  . AnotherOur study results reconfirmed that using SU to estimate 24-h UNa, the K-method, I-method, and T-method were inadequate at the Chinese population level. Our validation and assessment in Zhejiang Province showed a low accuracy for these common methods. One of the important reasons for this low accuracy may be the dietary lifestyle difference. In general, the K-method and T-method were based on the Japanese population, and the I-method was based on the European and North American population. In brief, these methods were not specifically established for the Chinese population. When these formulas were applied in the Chinese population, the bias is foreseeable. Moreover, the K-method was established using a second morning urine sample. Considering the large sample-size investigation convenience, in our study, we just collected the first morning urine instead of the second morning urine to estimate 24-h UNa. Therefore, validation studies in different populations, especially in the Chinese population, would be required. However, it might be uneasy to establish this kind of method to estimate population mean sodium intake under diverse dietary lifestyle available among nationwide China.r = 0.64, and the bias was 0.65 [Our study found that the correlation coefficient for the 24-h Na/K ratio and SU Na/K ratio was 0.43, and the bias between the Na/K ratio of 24-h urine and spot urinary Na/K ratio by the Bland–Altman method was −0.22. The correlation coefficient in East Asian individuals from the INTERSALT study was was 0.65 . The reawas 0.65 . Althougwas 0.65 , 12. TheHigh dietary sodium and low dietary potassium intakes still exists over the past four decades in China . ReducinTo our knowledge, one of the major strengths of our study is a relatively large sample of Chinese adults to assess and validate the three methods and to estimate 24-h UNa using SU samples (as a first assessment to estimate 24-h UK and the 24-h Na/K ratio). Nevertheless, our study had several limitations. First, a single 24-h urine sample was not sufficient to assess the between- and within-individual variation for individual sodium and potassium intakes. The precision might be improved by collecting multiple 24-h urine samples per person . HoweverThese methods to estimate 24-h UNa and UK from SU were inadequate at the population level in Zhejiang Province; however, the K-method showed the least bias among these methods. The SU Na/K ratio may be a useful and alternative method to 24-h urine collection for estimation of the urinary Na/K ratio in the Chinese population.24-h urine collection is essential for assessing UNa and UK but this is very inconvenient in large epidemiological surveys.24-h UNa and UK estimation equations based on SU have been developed but validity of these equations in Chinese population is still uncertain.The accuracy of estimating 24-h urinary Na/K ratio from spot urinary Na/K ratio in the Chinese population in unknown.Large sample size of Chinese adults to validate three methods in estimating 24-h UNa and UK using SU.These methods for estimating the 24-h UNa and UK from SU were inadequate.The spot urinary Na/K ratio may be a useful and alternative method for 24-h urine collection for the estimation of the urinary Na/K ratio in the Chinese population.supplementary material


While anti-PDL1 could target PD-L1 protein expressed on surface of tumor cells to the immune checkpoint blockade characteristic for tumor immunotherapy. This strategy could not only directly kill tumor cells but also improve CD8+ T cell level and facilitate effector cytokines release. In conclusion, the rational-designed PHDM+anti-PDL1 therapy strategy creates a new way for tumor immune-chemotherapy.The application of combinational therapy breaks the limitation of monotherapy and achieves better clinical benefit for tumor therapy. Herein, a hyaluronic acid/Pluronic F68-based copolymer-mixed micelle was constructed for targeted delivery of chemotherapeutical agent docetaxel (PHDM) in combination with programmed cell death ligand-1(PD-L1) antibody. When PHDM+anti-PDL1 was injected into the blood system, PHDM could accumulate into tumor sites and target tumor cells  Meanwhile, F68 was introduced into the copolymer micelle and the prepared mixed micelle to enhance otherapy . This stHA  was purchased from Bloomage Freda Biopharm Co. Ltd . 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS) was purchased from Shanghai Civi Chemical Technology Co., Ltd. . VES was obtained from Jiangsu XiXin Vitamin Co., Ltd. . Pluronic F68 was purchased from Sigma Chemical Co., Ltd. . DTX was acquired from Shanghai Jinhe Biotech Co., Ltd. . Dialysis bag  was obtained from Union Carbide Corporation. . Dulbecco's Modified Eagle's Medium (DMEM) was purchased from Thermo Fisher Scientific Inc. . Fetal bovine serum (FBS) was acquired from Tianjin Kangyuan Biotechnology Co., Ltd. (Tianjin China). Cell-counting kit-8 (CCK-8) was obtained from Nanjing KeyGen Biotech Co., Ltd. .2 atmosphere.B16-F10 cells were obtained from American Type Culture Collection  and cultured in DMEM with 10% FBS and 1% penicillin-streptomycin (Gibco) at 37°C under 5% CO7 cells in 75 μL of PBS) was subcutaneously injected into the left flank of mice, and when the tumor volume reached ~100 mm3, they were divided into several groups for further use. All the animal experiments were carried out according to the guidelines of the Experimental Animal Administrative Committee of Jiamusi University.C57BL/6 mice (6–8 weeks) and BALB/c mice were purchased from Beijing HFK Bioscience Co., Ltd. (China), and B16 cell suspension (about 2 × 10HA (0.02 mmol) was dissolved in 10 mL formamide at 50°C to complete dissolved and cooled to room temperature, subsequently, EDC (0.22 mmol), DMAP (0.22 m mol), and VES (0.2 mmol) were added into the mixture, and the mixture was stirred at room temperature overnight. The mixture was dialyzed by a dialysis bag  for 48 h. Finally, the solution was filtered, and lyophilized and HA-VES was obtained. .−8 mol L−1 was transferred into a brown flask, and a gradient concentration of HA-VES was added into each flask. Samples were under ultrasound in the water bath at 50°C for 2 h and stored overnight under dark conditions. Finally, the samples were measured by fluorescent spectrometer at 338 and 334 nm.Critical micelle concentration (CMC) of HA-VES was measured by pyrene fluorescent probe. Briefly, pyrene was 6 × 10The dialysis method was used to prepare DTX-loaded micelles. Briefly, 10 mg HA-VES/7 mg HA-VES + 3 mg F68 and 1 mg DTX were dissolved in DMSO and stirred at room temperature until completely dissolved. The mixture was transferred into a dialysis bag for dialysis and to remove organic solvent. Finally, the samples were filtered, and HDM and PHDM were prepared.ζ-potential of the HDM or PHDM were measured by Zetasizer . The morphology of different formulations was observed using transmission electron microscopy .The size and In-vitro stability measurement was investigated, and particle size change of different formulations was measured. Briefly, both formulations (5 mL) were placed in shaking incubator at 37°C, 100 rpm. At an interval time point, 0.5 mL samples were taken for particle size determination. Meanwhile, encapsulation efficacy  change was also measured by ultracentrifugation method. HDM or PHDM were dissolved in PBS (10 mL). The solution was centrifuged for 10 min, the filtrate (0.5 mL) was transferred into a 10-mL brown volumetric flask, and made up to the volume with methanol as the free DTX. Next, another unfiltered formulation was transferred into a 10-mL brown volumetric flask, and made up to the volume with methanol as the the total DTX, and calculate EE% as reference reported. For the stability assay, both formulations were placed in shaking incubator at 37°C. At an interval time point, 0.5 mL samples were taken for EE% measurement and particle size determination.in-vitro release process of different formulations was examined using the dialysis method. Different formulations were transferred into dialysis bags , and the dialysis bags were immersed in a 100 mL phosphate buffer saline  with moderate shaking (50 rpm) at 37 ± 0.5°C. Fresh medium was used to replace released medium at the predetermined time points. The cumulative release was plotted against time.The 4 cells and treated with various samples. At specified time intervals, 10 μL MTT solution was added into each well, and the plate was incubated for another 4 h. subsequently, the medium was removed and DMSO was added into each well. The plates were shaken and determine the absorbance at 450 nm was taken by the microplate reader.Cytotoxicity of different micelles was studied by the MTT method. In brief, B16 cells were seeded in 96 well plates at a density of 10The cell apoptosis assay was investigated with the Annexin V-FITC kit. The detailed operation procedure was according to the manufacturer of the kit.Nile red (NR) was used as a fluorescence probe to represent DTX and incorporated it into different formulations for a cellular uptake assay. Briefly, cells were seeded into a glass-covered six-plate well and allow attachment. Different formulations were added into each well and incubated for different times. At the interval time point, the medium was removed and washed with PBS, harvested with trypsin for flow cytometry, and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for Confocal laser scanning microscopy (CLSM) assay.For investigating intracellular drug release, LysoTracker-Green was used to pre-stain intracellular endo/lysosomes. Subsequently, different formulations were added into each well and incubated for 4 h. The cells were fixed with formalin and stained nucleic with DAPI, visualized using CLSM.in-vivo tumor-targeting of different formulations, DiR was used as fluorescence probe and incorporated into the micelles. Tumor-bearing nude mice were intravenously administrated different DiR-loaded micelles for circulation. At the interval time point, the mice were anaesthetized and visualized using an IVIS system. After 24 h, mice were sacrificed, and their major organs were obtained for imaging.To investigate B16 bearing mice were randomly divided for several groups . All mice received intravenous administrated of different formulations every other day for four times and measured for the tumor volume and body weight change. At the end of the trail, tumor tissues were weighed, and blood was collected for further study.Serum TNF-α,IFN-γ,and IL-2 level were measured using an ELISA kit. The detailed procedure was according to the manufacture of the kit.+ T cells. CD4-FITC and Foxp3-PE were used to label Tregs.Freshly isolated tumor tissue was obtained and cut into small pieces. The tumors were digested with collagenase IV for 1 h. Subsequently, the cells were filtered to prepare a single-cell suspension. The cells were stained with a cocktail antibody for flow cytometry. CD3-FITC and CD8-PE were used to detect CD8t-test.All data expressed as mean ± standard deviations (SD). Significant different between two groups was analyzed using two-tailed Student's via the ester reaction. Due to the presence of amount of hydroxyl groups in HA, it's easy to modify different functional backbones on the surface of HA through the ester reaction. Due to active carboxyl groups in VES and hydrophobic characteristic, it is suitable to construct HA-VES-grated amphiphilic copolymers, which could form micelle structures in an aqueous environment. The synthesis route and 1H NMR spectrum is illustrated in HA-VES was synthesized firstly in vitro and in vivo (Patil et al., −1, which indicated that HA-VES could form micelles at extremely low concentrations. The relative low CMC value indicated a better structural stability, which could possess the integrity of the copolymer micelle in extreme dilution. Particle size and ζ-potential of different formulations were measured, as shown in in-vitro stability of different formulations was measured, as shown in In-vitro drug release profiles were investigated, and the result is shown in CMC is an important parameter for evaluating the aggregation capability of copolymer in aqueous condition, which influences the self-assembly ability and structural stability of copolymer micelles both In-vitro cell cytotoxicity of different formulations were measured using MTT method against the B16 cell line. As shown in via passive diffusion, and the efficacy was limited. Meanwhile, no significant difference could be observed for both HDM and PHDM. Although F68 would form a hydrophilic shell on the surface of micelle, this shell was incomplete and could not totally block HA expose, only minor percentage of active targeting moiety could reach tumor-active targetability, which indicated F68 didn't influence the interaction between the CD44 receptor and HA residues.Cell apoptosis was considered as an important symbol of programmed cell death, and it is necessary to investigate whether the cell cytotoxicity arose from apoptosis or necrosis. As shown in via the CD44 receptor-mediated endocytosis. Flow cytometry results (CLSM and flow cytometry were both used for cellular uptake assay. As shown in  results  possesseIn order to investigate intracellular drug release behavior, LysoTracker Green was used to stain endo/lysosomes, and different formulations were incubated with cells. As shown in In-vivo biodistribution and antitumor activity of different formulations were investigated using tumor-bearing mice. DiR was used as fluorescence probe to detect the biodistribution of different formulations. As shown in In-vivo antitumor activity of different formulations was investigated using B16-bearing C57BL/6 mice. As shown in via the EPR effect and target tumor cell via HA-mediated endocytosis. PHDM + anti-PDL1 possessed the best antitumor activity, and this result was mainly because the introduction of the antibody could not only activate immune checkpoint-based immunotherapy, but also enhance the sensitivity of DTX-based chemotherapy. For the body weight study, only DTX possessed significant body weight loss, which was mainly attributed to the non-specific distribution and severe side effects of free DTX.In order to further demonstrate whether PHDM+anti-PDL1 achieve tumor immune-chemotherapy, a series of T cell cytokines was measured. As shown in + T cell and regulatory T cells were both measured by flow cytometry. As shown in +CD8+ cells increased, CD4+Foxp3+ cells decreased, and the ratio of CD8+/Treg increased for PHDM+anti-PDL1. It is well-acknowledged that tumor tissue possessed an immunosuppressive microenvironment and inactive effector T cells from PD-1/PD-L1 axis, which is the main reason for the failure of tumor therapy. Although a chemotherapy-based drug-delivery system could directly kill tumor cells via cytotoxic drugs, the clinical benefit is unsatisfied. In this study, the combination of chemotherapy and PD-L1 antibody is a suitable strategy for better antitumor therapy. The PD-L1 antibody could block the PD-L1 protein in tumor cells, which sends the “don't eat me” signal to T cells and induces a T-cell-based antitumor immune response. The released effector cytokines could enhance this process for long-term tumor progression inhibition. Meanwhile, PHDM could efficiently target tumor cells and intracellularly deliver antitumor molecules for tumor killing. All these results indicated that the combination therapy strategy could efficiently activate CD8+ T cells and decrease Treg to relieve tumor immunosuppression and activate the antitumor immune response.Subsequently, CD8Overall, in this study we constructed a DTX-loaded HA-based copolymer-mixed micelles in combination with PD-L1 antibody for tumor immune-chemotherapy. The as-synthesized HA-VES could be efficiently entrapped hydrophobic molecule DTX, and F68 could be used for mixed micelle construction. After injection, PHDM could efficiently accumulate into tumor sites and target tumor cells for tumor chemotherapy. While the PD-L1 antibody could also bind tumor cells for immune checkpoint inhibition for tumor immunotherapy, all the results demonstrated that PHDM+anti-PDL1 was a potential for tumor immune-chemotherapy.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Experimental Animal Administrative Committee of Jiamusi University.CZ and XD: perform the experiment and write manuscript. CS and SC: performance the data analysis. TC and DZ: perform data analysis. XZ and CY: contribute to the conception of the study. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Catharanthus roseus plants were graft-infected with both strains, either simultaneously or sequentially. Different combinations of lateral and apical grafting were applied to avoid possible benefits due to graft position. The infection was monitored for four months through a new diagnostic protocol developed for differentiation and relative quantification of the two strains. Regardless of the temporal or spatial advantage at grafting, FD-C generally outcompeted FD-D. The prevalence of FD-C increased over time and, at the end of the experiment, FD-C was the unique strain detected in the aerial part and the roots of 74% and 90% of grafted plants, respectively. These data indicate that the interaction between the two strains results in competitive exclusion. Understanding the bases of the competition between FD-C and FD-D may contribute to explain the biology of the coexistence of different FDp strains under field conditions, aiming at identifying potential suppressor strains, which can provide alternative and environmentally sustainable solutions for FD control.Flavescence dorée phytoplasmas  are plant pathogenic non-cultivable bacteria associated with a severe grapevine disease. The incidence of the two reference strains on cultivated grapevines is unbalanced, and mixed infections are rare. To investigate the interaction between the two strains,  Scaphoideus titanus Ball in a persistent and propagative way [Candidatus Phytoplasma vitis’ species [S. titanus [Arabidopsis thaliana hosts. Genetic characterization of FDp populations in a wine-growing region in Italy revealed that the -D subgroup was present in more than 70% of the disease cases, and the incidence of the -C strain was higher in plants than in insects [Flavescence dorée (FD) of grapevine, is a quarantine disease in Europe caused by the FD phytoplasma (FDp), a wall-less and uncultivable mollicute transmitted mainly by the monovoltine leafhopper tive way ,2. The vtive way , while ttive way . Outbreative way . The FDptive way ,7,8,9, o species , which h titanus , and sha insects . Moreove insects . In natu insects . Surpris insects ,15,16. D insects . Aim of  insects . Analogo insects .S. titanus as well as other occasional phloem feeder vectors [Management of FD epidemics relies on the understanding of the epidemiological cycle of the disease, involving the ampelophagous  vectors ,20,21, a vectors ,22. The nrdF), the maltose ABC transporter membrane subunit , and the group II intron gene (contig 12). These sequences were screened as targets for the development of the new diagnostic protocol based on the discrimination of the melting peak of the amplicons from each FDp isolate. The malG gene showed about 99% sequence identity between FD-C and the most frequent FD-D genotypes, including the malG1 of the FD-D isolate used in this work. The nrdF gene showed 98% sequence identity between -C and -D phytoplasmas, corresponding to 21 SNPs. The contig 12 showed 98.6% sequence identity between FD-C and -D strains (8 SNPs and 2 insertions/deletions). Primer pairs were designed on each of the three genes to include most of the SNPs between the two strains, as detailed in nrdF gene was developed. A single primer pair (NrdF_F28/NrdF_R121), matching both FD-C and -D sequences, and two strain-specific TaqMan probes were designed on a portion displaying two SNPs between the two strains  88.5% and 65.4% of the inoculated plants showed symptoms and tested PCR positive for FD-C and for FD-D respectively. The remaining FD-C and FD-D grafted plants were asymptomatic and PCR negative. The average phytoplasma load was similar between the two treatments and ranged between 1.24 × 103 and 2.27 × 105 GU/ng of total plant DNA (3 (standard error of the mean (SEM) ± 5.56 × 103), and it was significantly lower than that of FD-Dp (2.38 × 104 ± 3.57 × 104) (p < 0.05)  . No difft-test; p = 0.212) ranging between 2.32 × 103 and 1.69 × 106 GU/ng plant DNA , and the measured phytoplasmas loads were similar (lant DNA .p < 0.05)  .Time course experiments were designed to study the competition between FD-C and FD-D phytoplasmas in colonizing the aerial plant tissues. In the first experimental setting, the two FDp strains were inoculated contemporarily. Two combinations of double grafts were produced to exclude the possibility of an infection advantage due to the apical (A) or lateral (L) graft position a. At 60 In the second experimental setting, a temporal advantage of 15 days was provided to both the FDp strains. Four graft combinations were set up see b. When FMultiple infections of pathogen genotypes or species in the same pathosystem can drive disease dynamics and pathogen evolution ,24. ThisCatharanthus roseus as a model plant was established to shed light on the Flavescence dorée phytoplasma population dynamics observed in infected vineyards of Piedmont [Different factors, such as plant genetics, environmental variation, and intermediate hosts, can shape the prevalence of co-infection in space and time: combining field surveys and experimental approaches may help to unveil the role of each actor of such a complex scenario. In this work, an experimental design using Piedmont . The twoVicia faba after vector-mediated inoculation of the aerial part by Euscelidius variegatus [Ca. P. asteris’ [Ca. P. asteris’ strains, severe (SAY) and dwarf (DAY), showed instead different infection dynamics; however, the more aggressive behavior of the SAY strain could be a combination of genetic differences in the effector machinery of the two isolates and a possible imbalance in the starting inoculum of the two strains [On single infection, FD-C and FD-D strains showed similar virulence, symptomatology, and infection dynamics. Moreover, the time course experiments revealed that the speed and efficiency of colonization of both shoots and roots were comparable. Both phytoplasma strains reached the root system within 12 days from grafting, then spread to the aerial part of the plant, where they could be detected after 45 days together with the first symptom appearance. Indeed, FDp rapidly infects also the root system of the experimental host riegatus . Rapid masteris’ . The two strains . In our  strains . Similar strains .C. roseus with temporal advantage seems to rule out a mechanism of competition based on “first come first served” since FD-C strain outgrew FD-D even when grafted afterwards. This phenomenon was particularly evident in the roots. However, interference competition may still be a possible mode of relationship between FD-C and FD-D phytoplasmas. Interference competition among parasites may be based either on the occupation of the available space (priority) or the release of growth-inhibiting compounds  [Ca. P. mali’ together with the antigenic membrane protein (Amp) of ‘Ca. P. asteris’ bind actin filaments of plant and insect cells, respectively [Ca. P. mali’, mild strain suppression of aggressive strains is mediated by the AP460 gene, encoding an AAA+ ATPase, a group of proteins that compose the T6SS system [ftsH genes are described in the FDp genome, with a role in host adaptation [The prevalence of FD-C phytoplasma indicates that the interaction between the two strains results in competitive exclusion. Three mechanisms may explain competition between parasites within a host: exploitation, apparent, and interference competition ,30,31. Flopathy) . In the lopathy)  and the ectively ,39,40. Tectively  and it bectively , among wectively . FD-C anectively . Strain-ectively ) suggestectively ,46,47, cS system . Notablyaptation .C. roseus results in FD-C phytoplasma dominance, in Piedmontese vineyards  the predominant strain is FD-D [Scaphoideus titanus is the vector of FDp. The competitive strategy successful in the plant host may be unsuccessful in the insect one. It is worth noting that the incidence of FD-C strain, even though low in general in the vineyards, is higher in plants than in insects [V. vinifera and C. roseus) cannot be excluded. The effects of the two FDp strains competing within the vector are currently under analyses, with special regard to colonization of the insect salivary glands. The draft status of FD-C and -D genomes [While strain competition in  is FD-D . Phytopl is FD-D , and the insects . This ev insects . In any  genomes ,52 does Ca. P. mali’ two mild strains suppress the infection of the aggressive ones both in C. roseus and apple trees [FD is a severe quarantine disease and a threat to vineyard productivity in the most important European wine-producing areas. Control measures are mainly based on large-scale insecticide treatments, with detrimental impacts on public health and benign insects, roguing of infected plants and use of healthy plant material. Therefore, alternative and environmentally sustainable solutions are needed. Unveiling the mechanisms of FDp strain adaptation to different hosts may identify new targets to broaden our control strategies. A better understanding of the molecular bases of the competition between FDp strains may identify mild suppressor strains to outcompete the severe ones. In the case of ‘le trees , and arele trees . Recoverle trees , metabolle trees  and epigle trees  of the rle trees , and thele trees ,12. The Catharanthus roseus (L.)) by grafting. Healthy periwinkle plants were obtained by seed and grown in pots in a greenhouse under controlled conditions . For each experiment, periwinkle plants were graft-inoculated and sampled at 30 days post grafting (dpg) to confirm their infection status and to quantify the phytoplasma load. Infected plants with phytoplasma loads between 1.04 × 104 and 4.32 × 104 GU/ng plant DNA were selected as scion donors for the grafting experiments detailed below.The Flavescence dorée 16SrV-C  phytoplasma strain (FD-Piedmont) was isolated in Italy ,57. The Time course experiments were designed to study the ability of the two strains to move systemically through the aerial part of the plant and the root system following localized grafting.Aerial part. Twelve weeks old periwinkles (26 plants) were graft-inoculated with either FD-C or FD-D phytoplasma strains. After 60 days from the grafting, three leaves of each plant were sampled and analyzed by Real Time PCR to determine phytoplasma load.Roots. Twelve weeks old periwinkles (15 plants) were graft-inoculated with either FD-C or FD-D phytoplasma strains. After 3, 7 and 12 days starting from the grafting, the roots of three plants were sampled and analyzed until FDp was detectable in at least one individual. To investigate the effects of the two FDp strains on periwinkle root growth, nine plants were graft-infected with either FD-C or FD-D phytoplasmas, together with nine plants grafted with healthy scions. At 120 dpg, leaf samples were collected from each grafted plant to confirm the infection status. At the same time, roots were also collected, and their fresh weight was measured.Two time course experiments were designed to study the competition between FD-C and FD-D phytoplasmas in colonizing the aerial plant tissues. In the first experimental setting, the two FDp strains were grafted contemporarily. Two combinations of double grafts were produced to exclude the possibility of an infection advantage due to the apical (A) or lateral (L) graft position. In particular, two treatments were applied: lateral grafting with FD-Cp and apical with FD-Dp , and vice versa  a. For toLeaf samples were collected from the top of three branches of each graft-inoculated plant at different sampling times as described above. Total nucleic acids were extracted from 0.5 g of leaves and petioles according to Pellettier et al. . PrecipinrdF; partial: MK091399, MK091398 [malG; partial: MH547713, MH547714 [Three FDp genomic regions were screened as targets for the development of the new diagnostic and quantification protocol based on the discrimination of the melting peak analyses of the amplicons from each FDp isolate: the ribonucleoside-diphosphate reductase 2 β subunit , the maMH547714 ; full leMH547714 ), and thMH547714 ; Xavier MH547714 ). PrimernrdF genes. The primers NrdF_F28 and NrdF_R121 were designed between positions 28 and 121 after the gene starting codon and they matched both FD-C and -D sequences. On the amplified sequence, two specific TaqMan probes for nrdF -C or -D were designed on a portion displaying two SNPs between FD-C and FD-D phytoplasma strains  on a CFX-BioRad. Each sample was run in triplicate on the same plate. Cycling conditions were as follow: initial denaturation at 95 °C for 3 min, followed by 45 cycles of 10 s at 95 °C and 30 s at 60 °C. To obtain two standard plasmids for FDp quantification, nrdF-C and -D genes were amplified from total DNA extracted from infected periwinkles with the primers NrdF_F20 (5′-TGCATAAATTTCACGGAGCT-3′)/NrdF_R (5′-TAACGGACAAAAGCGTTTAC-3′) using standard PCR conditions. After purification of PCR products with the DNA Clean & Concentrator Kit (Zymo Research), the amplicons were ligated into the pGEM-T easy cloning vector following the manufacturer’s instructions . After transformation of E. coli DH5α competent cells by heat shock, recombinant plasmids were extracted using the Wizard SV Plus Minipreps DNA Purification System  and quantified with NanoDrop 2000 TM Spectrophotometer . For absolute quantification of the two FDp strains, two standard curves were obtained from recombinant pGEM plasmids harboring a fragment of the corresponding targets , serially diluted from from 100 pg to 10 fg, corresponding to 107 to 10 genomic units (GU) of FDp. Standard curves were constructed with the CFX Manager™ Software, by linear regression analysis of the Cq value of each standard dilution replicate over the log of the number of plasmid copies present in each sample. Moreover, to verify the reliability of the probes in discriminating between the two strains when present simultaneously in the DNA sample, the two standard plasmids were mixed at different concentration ratio  that were used as template for the qPCR.For the development of a quantitative strain-specific protocol based on TaqMan chemistry, a duplex Real Time PCR was designed on  strains . SpecifiPAST software was adopted for statistical analysis . Analysi


The cognitive function of Bean-PS was assessed using the Morris water maze (MWM) test and a passive avoidance task (PAT). The expression of acetylcholine transferase (ChAT) and acetylcholinesterase (AchE) in the hippocampus was assessed via immunohistochemistry. A positron emission tomography (PET) scan was used to measure the glucose uptake in the rat brain. Results: Treatment with Bean-PS enhanced memory function in the Morris water maze (MWM) test. Consistent with the behavioral results, treatment with Bean-PS diminished the damage to cholinergic cells in the hippocampus, in contrast to those of the TMT group. The TMT+Bean-PS group showed elevated glucose uptake in the frontal lobe of the rat brain. Conclusion: These results demonstrate that Bean-PS protects against TMT-induced learning and memory impairment. As such, Bean-PS represents a potential treatment for neurodegenerative disorders, such as Alzheimer’s disease.Background: Trimethyltin (TMT) is a potent neurotoxin affecting various regions of the central nervous system, including the neocortex, the cerebellum, and the hippocampus. Phosphatidylserine (PS) is a membrane phospholipid, which is vital to brain cells. We analyzed the neuroprotective effects of soybean-derived phosphatidylserine (Bean-PS) on cognitive function, changes in the central cholinergic systems, and neural activity in TMT-induced memory deficits in a rat model. Methods: The rats were randomly divided into an untreated normal group, a TMT group (injected with TMT + vehicle), and a group injected with TMT + Bean-PS. The rats were treated with 10% hexane (TMT group) or TMT + Bean-PS (50 mg·kg Trimethyltin (TMT) intoxication is regarded as an appropriate model of chronic neuronal degeneration associated with cognitive impairment, and is therefore useful in the study of Alzheimer’s disease (AD) ,2. The oSeveral studies have shown that the cognitive dysfunction was associated with damaged cholinergic neurons in the brains of animal models of AD. Damage to the cholinergic system in the brain is closely associated with memory deficits. These anatomical and behavioral findings in TMT-intoxicated rats have been used to develop an attractive model of degenerative diseases such as AD . AD has Phosphatidylserine (PS) is the major anionic phospholipid found in the inner leaflet of eukaryotic cell membranes. PS-supplemented rodents showed enhanced memory, learning capacity, and other cognitive parameters . In humaHowever, few studies have reported the effects of soybean-derived phosphatidylserine (Bean-PS) on cognitive improvement and its underlying mechanisms. The purpose of this study is to investigate whether Bean-PS prevents the neurodegeneration of the hippocampus, and the impairment of learning and memory, induced by TMT. The study demonstrated that Bean-PS improved cognitive function, and activated cholinergic systems in the hippocampus in addition to neural activation, in rats with TMT-induced cognitive deficits.p < 0.001, on Days 1 and 4, ** p < 0.01, on Days 2 and 3). The TMT + Bean-PS group demonstrated amelioration of spatial memory and learning ability relative to the TMT group starting from Day 2 (p < 0.05) and Day 4 (p < 0.05). In the retention test, the times spent on the platform varied significantly among the groups. The TMT group spent less time on the platform than the normal group . The TMT + Bean-PS group did not affect the time spent in the platform area as seen in The passive avoidance test was conducted to determine the ability of working memory and learning. As shown in 2, 18 = 8.53, p < 0.01) and CA3 .The results of the evaluations of the ChAT-positive cells per section from the different hippocampal formations are shown in p < 0.05). Immunoreactivity of ChAT in the TMT + Bean-PS group was significantly increased in CA1 (p < 0.05), as seen in p < 0.001). In particular, there were significant differences in both CA1  and CA3 . However, the AChE reactivity in the TMT+Bean-PS group was higher than that in the TMT group, particularly in CA1 (p < 0.01) and CA3 (p < 0.01).The numbers of ChAT-positive cells in the TMT+Bean-PS group were higher than those in the TMT group, particularly in CA1 (18F-fluorodeoxyglucose (FDG) uptake measurements from the different brain regions. The cerebral glucose activity of the TMT group was markedly reduced in the hippocampus and frontal lobe compared with that of the normal group in statistical parametric mapping (SPM) analysis of FDG-positron emission tomography (PET) (p < 0.05). In addition, the activity of the TMT + Bean-PS group was significantly increased in the frontal lobe and hippocampus compared with the TMT group (p < 0.05).AD is characterized by deterioration in memory, thinking, and the ability to carry out activities. An estimated 50 million people worldwide manifest dementia, and almost 10 million new cases are reported every year . There iTMT exposure triggers severe behavioral and cognitive defects in both humans  and expeOur study indicated that spatial memory impairment was ameliorated in the TMT+Bean-PS groups during the training days in contrast with that of the TMT group. In addition, the data demonstrated that Bean-PS preserved the TMT-induced reduction in spatial retention. These results suggested that Bean-PS improved learning and memory deficits in TMT-intoxicated rats used as experimental models of neurodegeneration for the study of Alzheimer-like diseases ,39.The animal model used in this study clearly demonstrates the functional significance of hippocampal neurodegeneration induced by TMT. The cholinergic system is involved in information processing associated with hippocampal learning and memory . The hipBased on the cholinergic hypothesis, patients with senile dementia show a selective and irreversible lack of cholinergic function in the brain . TherefoIt is quite probable that the observed improvement in spatial learning deficits following the treatment with Bean-PS of rats was related to the enhanced release of ACh. According to the results from the Morris water maze (MWM), exposure to Bean-PS ameliorated the TMT-induced deficits in learning and memory. In addition, treatment with Bean-PS decreased cell loss, increased central cholinergic function, and prevented degeneration of cholinergic neurons mediating cognitive processes .Our previous study reported that treatment with Bean-PS dissolved in medium-chain triglyceride (MCT) improved cognitive function and enhanced the neural activity in rats with TMT-induced learning and memory deficits. The active biological ingredient in the resulting extract is affected by the solvents . TherefoThe present study analyzed the effect of 10% hexane solvent on Bean-PS-attenuated TMT-induced cognitive defects in the Morris water maze, and found a protective effect in contrast to TMT-induced reduction in ChAT and AchE in the hippocampal areas.Exposure to Bean-PS resulted in upregulation of glucose uptake in the hippocampus and frontal lobe. Administration of Bean-PS may have robust therapeutic potential as a treatment for neurodegenerative disorders, such as AD.n = 9); TMT injected with vehicle ; and TMT injected along with 50 mg/kg−1 Bean-PS . The rats were injected intraperitoneally (i.p.) with TMT  dissolved in 0.9% saline and then returned to their home cages. The TMT+Bean-PS mixture ) was dissolved in 10% hexane and orally administered for two weeks after TMT-induced neurodegeneration.Male Sprague-Dawley rats weighing 250–580 g each were purchased from Samtaco Animal Corp. All rats were housed individually in a room at 23 °C (room temperature) under an alternating 12 h light/dark cycle. The rats were fed a commercial diet and provided with tap water ad libitum throughout the study. Food and water were made accessible ad libitum. This experiment was conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, revised in 1996, and was approved by the Institutional Animal Care and Use Committee of Kyung Hee University (KHUAP(SE)-18-073, 05/30/2018). In this study, the rats were randomly assigned to three groups: untreated, naïve , 2% phosphatidylcholine (PC), and 6% phosphatidic acid (PA). The Bean-PS contained palmitic (17.7%), palmitoleic (1.3%), stearic (1.3%), oleic (14.4%), limoleic (61.2%), linolenic (1.4%), eicosapentaenoic acid (EPA), and other fatty acids (9.4%). PS and 10% hexane, used as a solvent for PS, were provided by Doosan Co. Glonet BG  and the PS was stored in a freezer (−60 °C).From the 17th day after the treatment with Bean-PS, the Morris water maze test was performed for 5 days. The black, plastic pool used in the Morris water maze test measured 200 cm in diameter and was filled to a depth of 35 cm with clear water maintained at 23 ± 2 °C. It was inserted in a submerged platform using external cues around the pool and within the behavioral room. For behavioral analysis, a personal computer was used to assort a charge coupled device (CCD). A probe test was run after the last trial on Day 4. In the experiment, the rats were exposed to an acquisition trial on each of 4 consecutive days. On Day 5, they were also trained in retention tests. The rats were allowed to search for the platform for 180 s during the acquisition test. On Day 5 of the retention test, the rats underwent a 1 min probe trial, in which the platform was eliminated from the pool. Probe trials were run at 1 min intervals. The animals in the performance test of each water maze trial were evaluated with a personal computer during the behavioral analysis .A passive avoidance task was performed after 21 days of the treatment with Bean-PS. A test chamber was composed of two platforms, one light  and the other dark . A guillotine door opening (6 cm × 6 cm) was made on the floor in the center of the divider between the two partitions. Stainless steel bars, of diameter approximately 5 mm, were spaced at about 1 cm apart. Electric shocks  were transferred to the grid on the floor of the dark compartment. All rats were again located on the platform, and the escape latency was recorded in the retention test.18F fluorodeoxyglucose (FDG), rats were anesthetized with sodium pentobarbital . Rats were perfused with saline solution, followed by 4% paraformaldehyde (PFA). The brain was anatomized from the skull, post-fixed in PFA overnight, and stored in a 30% sucrose in PBS until it subsided. The brain was embedded and serially sectioned on a cryostat  at 30 μm thickness in the coronal plane and the sections were collected in phosphate buffered saline (PBS). The primary antibodies against the following specific antigens were used: ChAT and AchE. The primary antibody was diluted from the concentrate with the blocking solution , 2% blocking serum in PBST). The primary antibody was infused into the brain sections for 72 h at 4 °C. After several rinses in PBST, the sections were incubated with secondary biotinylated antibodies against rabbit immunoglobulin G (IgG) or sheet IgG  for 2 h. After washing with PBST, the sections were incubated with an appropriate biotinylated secondary antibody and processed with an avidin–biotin complex kit . The staining was conducted using 0.05% 3,3′-Diaminobenzidine (DAB) in the presence of 0.003% H2O2 in 0.1 M PB. After rinsing with 0.1 M PB, the stained tissue sections were mounted on the slide. The images were captured using a DP2-BSW imaging system  and processed using Adobe Photoshop Cells. Those testing positive for ChAT and AChE were counted on a grid that was placed on CA1 and CA3 in the hippocampus area. The number of cells was counted at a magnification of 100×, using a rectangular microscopic grid measuring 200 × 200 µm2. The cells were counted in 3 sections per rat within the hippocampus.After completion of the p < 0.05, K > 50).After completion of the behavioral test, animals underwent prior fasting over 12–15 h and bedding was changed during the fasting period to avoid ingestion of bedding. The animals were treated with 500 mCi/100 g 18F-FDG via tail intravenous insjsmsjection, and the animals inhaled 2% isoflurane in 100% oxygen  until the positron emission tomography (PET) scan. A transverse resolution of <1.8 mm was used at the center . The emit test for single comparisons, using Prism 5.01 software . In all statistical assessments, p < 0.05 was considered to be statistically significant. Results are expressed as the mean ± standard error of the mean.Results were assessed using one-way or two-way analysis of variance (ANOVA), and repeated measures of ANOVA, followed by Tukey’s post hoc test, using SPSS 15.0 for Windows, for statistical analysis, for multi-group comparisons, and Student’s Treatment with Bean-PS attenuated the TMT-induced memory impairment in the MWM test. Furthermore, the Bean-PS improved cognitive function, and generated a neuroprotective effect by increasing the expression of cholinergic neurons and glucose metabolism. Thus, Bean-PS is a potential agent that can be used to prevent and protect against neurodegenerative diseases such as dementia and AD. It may represent a good therapeutic agent for of the management of AD.